JP2018535208A - How to treat Angelman syndrome and related disorders - Google Patents

Abstract

  Provided herein are methods of treating Angelman syndrome and / or Prader-Willi syndrome, comprising administering an effective amount of a T-type calcium channel antagonist to a subject in need of treatment.

Description

This application claims the benefit of US Provisional Patent Application No. 62 / 245,038, filed Oct. 22, 2015, the entire disclosure of which is incorporated herein by reference.

TECHNICAL FIELD The present disclosure relates to the treatment of diseases by administering pharmaceutical compounds. Specifically, this disclosure relates to the treatment of Angelman syndrome and Prader-Willi syndrome by administering a T-type calcium channel antagonist.

  Angelman syndrome is a genetic disease caused by deletion or inactivation of a gene on maternally inherited chromosome 15 by silencing through imprinting of a paternal copy of the gene. The syndrome is characterized by intellectual and developmental disabilities, sleep disturbances, seizures, jerky behaviors, and a happy attitude with frequent laughter or smiles. The number of people affected by Angelman Syndrome is the same for males and females, with a disease rate of about 15,000th of the general population.

  Angelman syndrome is most commonly caused by a loss of a maternally derived region of chromosome 15, but also by a single parental disomy, translocation or single gene mutation in that region. In regions of the chromosome that are understood to be critical for Angelman syndrome, the expression of certain genes varies greatly depending on whether they are maternal or paternal, which is sex-specific through DNA methylation. Due to epigenetic imprinting. In normal individuals, the gene UBE3A maternal allele, which is part of the ubiquitin pathway, is expressed and the paternal allele is specifically silenced in the developing brain. Angelman syndrome occurs when maternal parts are lost or mutated. When the paternity-derived part is lost or mutated, a related syndrome (Prader-Willi syndrome) occurs.

  The most common genetic disorder leading to Angelman syndrome is a deletion of the approximately 4 megabase maternal portion of chromosomal region 15q11-13 resulting in a lack of expression of UBE3A in the imprinted paternal brain region. UBE3A encodes an E6-AP ubiquitin ligase. Mice that do not express maternal UBE3A exhibit severe dysfunction including a lack of hippocampal memory formation in a learning paradigm with hippocampal-dependent contextual fear conditioning. Maintenance of long-term synaptic plasticity in the hippocampal region CA1 is also inhibited in vitro.

  Symptoms of Angelman syndrome include functionally severe developmental delay, language dysfunction (minimum language use or language unavailability), movement and balance disorders (including gait ataxia and / or limb tremors) , Frequent laughter / smile, appearance-satisfactory attitude, personality that tends to excite (often exhibiting behavior that flutters hands), excessive physical activity, and lack of concentration. Frequently observed symptoms (80% or more cases) include disproportionate growth, seizures, and abnormal brain waves (EEG) with delayed head circumference. In EEG, ocular closure interrupted by the most noticeable very large amplitude 2-3 Hz rhythm in frontal guidance, symmetric 4-6 Hz high voltage rhythm, and spikes and sharp waves in occipital guidance Three different inter-seizure patterns of 3-6 Hz activity related to are seen in Angelman syndrome. Furthermore, about 20 to 80% of the symptoms associated with Angelman syndrome include occipital flatness, occipital artery groove, tongue protrusion, eating disorder, prognathia, wide mouth , Fluency, mouthing behavior, depigmentation, extremely active deep leg tendon reflexes, hypersensitivity to heat, abnormal sleep patterns, water sensitivity, abnormal feeding-related behaviors, obesity, spinal column Scoliosis and constipation are included. Spiders typically occur in 85% of patients with Angelman syndrome by 3 years of age, while less than 25% of patients show seizures by 1 year of age. Types of seizures include atypical absence, generalized tonic clonic seizures, tonic seizures and myoclonic seizures. Some patients exhibit more than one type of seizure.

  There is currently no cure for Angelman syndrome. Because Angelman syndrome can result in multiple types of seizures, it can be difficult to select an appropriate antispasmodic medication to treat epilepsy. Because Angelman syndrome affects sleep patterns, melatonin may be used to promote sleep. Gentle laxatives are often used to encourage regular bowel movements. In addition to drug administration, physical therapy is used to improve joint mobility and prevent joint stiffness. Speech and hearing therapy is commonly used to address communication problems.

  T-type calcium channels are low voltage activated calcium channels that open during membrane depolarization and mediate calcium influx into cells after action potentials or depolarization signals. T-type calcium channels, which are known to exist in the myocardium and smooth muscle, are also present in many neuronal cells in the central nervous system. T-type calcium channels (transient open calcium channels) are the ability to be activated by more negative membrane potential, their small single channel conductance, and conventional targeting of L-type calcium channels It differs from L-type calcium channels (long-acting calcium channels) due to their non-responsiveness to calcium channel antagonist drugs.

  T-type calcium channels open after a small membrane depolarization. T-type calcium channels have been studied mainly in relation to the functions of neurons and cardiomyocytes and are known to be associated with hyperexcitability disorders such as epilepsy and cardiac dysfunction. Voltage-gated calcium channels are generally not expressed in non-excitable cells, but there is evidence that T-type calcium channels are expressed in non-excitable lineage cancer cells.

  T-type calcium channels are activated and deactivated by small-scale membrane depolarization and show a slow deactivation rate. Thus, these channels carry depolarization currents at low membrane potentials and can mediate cellular “window” currents, which are activated and steady at low or resting membrane potentials. Occurs within the voltage overlap with state inactivation. T-type calcium channels can maintain the window current at unstimulated or resting membrane potential, thereby allowing sustained inward calcium flow through some channels that are not inactivated. Mediation of window current allows control of intracellular calcium levels by T-type calcium channels in both electrically ignited cells such as neurons and non-excitable tissues under unstimulated or resting cell conditions.

A voltage-gated calcium channel is composed of several subunits. The α ₁ subunit is the main subunit that forms a through-hole in the channel. The α ₁ subunit also determines the type of calcium channel. The β, α ₂ δ, and γ subunits present only in certain types of calcium channels are auxiliary subunits that play a second role in the channel. The α ₁ subunit is composed of four domains (I-IV), each domain contains six transmembrane segments (S1-S6), and the hydrophobic loop between the S5 and S6 segments of each domain defines the channel pore. Form. T-type calcium channel subtypes are defined by specific α ₁ subunits as shown in Table 1.

Ca ²⁺ / calmodulin-dependent protein kinase II (calmodulin kinase II or CaMKII) is a serine / threonine-specific protein kinase that is regulated by the Ca ²⁺ / calmodulin complex. CaMKII has 28 isoforms, is involved in many signaling cascades and may be a regulator of learning and memory, and homeostasis and reabsorption in cardiomyocytes, chloride ion transport in the epithelium, positive T cells May play a role in the selection and activation of CD8 T cells. Misregulation of CaMKII can lead to Alzheimer's disease, Angerman syndrome and cardiac arrhythmias. CaMKII is also associated with long-term potentiation (LTP), a molecular process that enhances activated synapses that may be involved in the memory process and is therefore considered important for memory formation.

  The sensitivity of the CaMKII enzyme to calcium and calmodulin depends on the variable and self-association domains. Initially, the enzyme is activated by binding to the calcium / calmodulin complex, which results in phosphorylation of the threonine 286 site and activation of the catalytic domain. Once activated, accumulation of calcium and calmodulin at high levels can result in autophosphorylation, which can result in sustained activation of the CaMKII enzyme. The accumulation of holoenzymes in the ring structure allows phosphorylation of adjacent CaMKII enzymes due to the close positional relationship of these rings, thereby enhancing autophosphorylation. Dephosphorylation of threonine 286 residue inactivates CaMKII.

  The present disclosure provides a method of treating Angelman syndrome or Prader-Willi syndrome. The method includes administering a therapeutically effective amount of a T-type calcium channel antagonist to a subject in need of such treatment. Also provided is the use of a T-type calcium channel antagonist for treating Angelman syndrome or Prader-Willi syndrome. The present disclosure also provides the use of a T-type calcium channel antagonist in the manufacture of a medicament for treating Angelman syndrome or Prader-Willi syndrome.

  In some embodiments, the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.

  In some embodiments, the T-type calcium channel antagonist is a small molecule.

  In some embodiments, the T-type calcium channel antagonist is an antibody.

  In some embodiments, the T-type calcium channel antagonist is an siRNA.

  In some embodiments, the T-type calcium channel antagonist selectively targets CaV3.1.

  In some embodiments, the T-type calcium channel antagonist selectively targets CaV3.2.

  In some embodiments, the T-type calcium channel antagonist selectively targets CaV3.3.

  In some embodiments, the T-type calcium channel antagonist antagonizes the T-type calcium channel in the cell when the membrane potential of the cell is in the range of about −60 mV to about −30 mV, eg, about −40 mV.

  In some embodiments, the T-type calcium channel antagonist is mibefradil, diltiazem, nifedipine, nitrendipine, nimodipine, nildipine, nigurdipine, nicardipine, nisoldipine, amlodipine, felodipine, isradipine, riocidin, galopamil, verapamil, thiapamyl, pimozide, thioridin, NNC55-0396, TTL-1177, anandamide, pimozide, penfluridol, clopimozide, fluspirylene, haloperidol, droperidol, benperidol, triperidol, melperone, lenperone, azaperone, domperidone, anthrafenine, aripiperazole, ciprofloxacin, dapiprazole, Dropropidine, etoperidone, itraconazole, ketoconazole, levo Lopropidin, mepiprazole, naphthopidil, nefazodone, niaprazine, oxypertin, posaconazole, trazodone, urapidil, vesnarinone, manidipine, nilvadipine, benidipine, efonidipine, flunarizine, anandamide, romeridine, phenytoin, zonisamide, U-9202l, roll-9202 NNC55-0396, TTA-A2, TTA-A8, TTA-P1, 4-aminomethyl-4-fluoropiperidine (TTA-P2), TTA-Q3, TTA-Q6, MK-5395, MK-6526, MK-8998 , Z941, Z944, ethosuximide, fence succinide, mesuximide, desmethylmethsuximide, efonidipine, trimethadione, Metajion, ABT-639, TTL-1177, KYSO5044, nickel and Kurutokishin (kurtoxin), and is selected from the group consisting of.

  In some embodiments, the T-type calcium channel antagonist is selected from the group consisting of mibefradil, efonidipine, TTL-1177 and nickel, and combinations thereof.

  In some embodiments, the T-type calcium channel antagonist is mibefradil.

  In some embodiments, the T-type calcium channel antagonist is MK-8998.

  In some embodiments, the disease is Angelman syndrome.

  In some embodiments, the disease is Prader-Willi syndrome.

  In some embodiments, the treatment includes reducing or ameliorating neurological symptoms that can be one or more of hyperactivity, abnormal sleep patterns, seizures and ataxia.

  In some embodiments, the treatment includes reducing the frequency of seizures in the subject.

  In some embodiments, the treatment includes reducing the severity of the subject's seizure.

  In some embodiments, the treatment includes reducing the frequency of dystonia in the subject.

  In some embodiments, the treatment comprises reducing the severity of the subject's dystonia.

  In some embodiments, the treatment includes reducing the frequency of the subject's ataxia.

  In some embodiments, the treatment includes reducing the severity of the subject's ataxia.

  In some embodiments, the treatment includes reducing the frequency of the subject's tremor (eg, tremor of the limb).

  In some embodiments, the treatment includes reducing the severity of the subject's tremor (eg, tremor of the limb).

  In some embodiments, the treatment includes reducing the severity of the subject's ataxia.

  In some embodiments, the treatment includes improving cognition or reducing cognitive impairment in the subject.

  In some embodiments, the treatment includes improving memory or reducing memory impairment in the subject.

  In some embodiments, the treatment includes improving attention or reducing attention deficit in the subject.

  In some embodiments, the treatment includes reducing obesity in the subject.

  In some embodiments, the treatment includes reducing the amount or rate of weight gain of the subject.

  In some embodiments, the treatment includes reducing the weight of the subject.

  In some embodiments, the treatment includes reducing a subject's feeling of hunger or increasing a feeling of fullness.

  In some embodiments, the selective T-type calcium channel modulator substantially crosses the blood brain barrier. In other embodiments, the T-type calcium channel modulator does not substantially cross the blood brain barrier.

  In some embodiments, treatment includes administering an additional therapeutic agent to the subject, which can be, for example, an additional T-type calcium channel inhibitor or an anticonvulsant.

  In some embodiments, the treatment includes performing additional therapy, which may be selected from the group consisting of, for example, physical therapy, occupational therapy, communication therapy, and behavioral therapy.

  The present disclosure provides a method of treating a disease or disorder involving CaMKII autophosphorylation. The method includes administering a therapeutically effective amount of a T-type calcium channel antagonist to a subject in need of such treatment. Also provided is the use of a T-type calcium channel antagonist to treat a disease or disorder involving CaMKII autophosphorylation. The present disclosure also provides the use of a T-type calcium channel antagonist in the manufacture of a medicament for treating a disease or disorder associated with CaMKII autophosphorylation.

  In some embodiments, the disease or disorder is Angelman syndrome or Prader-Willi syndrome.

  In some embodiments, the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.

  In some embodiments, the T-type calcium channel antagonist is a small molecule.

  In some embodiments, the T-type calcium channel antagonist is an antibody.

  In some embodiments, the T-type calcium channel antagonist is an siRNA.

In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.1.

In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.2.

In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.3.

  In some embodiments, the T-type calcium channel antagonist antagonizes the T-type calcium channel in the cell when the membrane potential of the cell is in the range of about −60 mV to about −30 mV, eg, about −40 mV.

  Also provided is a method of treating obesity in a subject in need thereof, comprising administering to the subject a T-type calcium channel antagonist.

  In some embodiments, the treatment includes reducing the weight of the subject.

  In some embodiments, the treatment includes reducing the amount or rate of weight gain of the subject.

  In some embodiments, the treatment includes reducing the weight of the subject.

  In some embodiments, the treatment includes reducing a subject's feeling of hunger or increasing a feeling of fullness.

  In some embodiments, the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.

  In some embodiments, the T-type calcium channel antagonist is a small molecule.

  In some embodiments, the T-type calcium channel antagonist is an antibody.

  In some embodiments, the T-type calcium channel antagonist is an siRNA.

In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.1.

In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.2.

In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.3.

  In some embodiments, the T-type calcium channel antagonist antagonizes the T-type calcium channel in the cell when the membrane potential of the cell is in the range of about −60 mV to about −30 mV, eg, about −40 mV.

  Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. If only the first page number of a reference is cited, it should be understood that the reference covers all pages of the reference. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

  Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Audiogenicity using UBE3 / 129sv mice treated with mibefradil (10 mg / kg, 40 mg / kg) or MK-8998 (also known as “CX-8998”) (10 mg / kg, 30 mg / kg, 60 mg / kg) It is a figure which shows the result of a habit test.

  The present disclosure describes that T-type voltage-gated calcium channels are associated with Angelman syndrome. The present disclosure further describes that modulation of T-type voltage-gated calcium channels may be effective for the treatment of related conditions such as Angelman syndrome and Prader-Willi syndrome.

Without being bound by any theory, T-type calcium channel antagonists such as mibefradil described herein are serine-threonine specific protein kinases that are regulated by the Ca ²⁺ / calmodulin complex and are involved in various signaling cascades. It can inhibit the inhibitory autophosphorylation of certain Ca ²⁺ / calmodulin-dependent protein kinase II (ie CaMKII). Neuronal depolarization increases the level of CaMKII autophosphorylation, and extracellular calcium chelation strongly reduces basal CaMKII autophosphorylation, raising the level of total CaMKII in the cytosolic fraction. Inhibition of T-type calcium channels by mibefradil (5 μM) or NiCl ₂ (100 μM) significantly reduced the threonine autophosphorylation of CaMKIIα by 37% or 35%, respectively, basal CaMKIIα activation and self It has been shown that it is suggested that phosphorylation is at least partially supported by calcium influx through T-type calcium channel inhibitors. Pasek et al., Mol. Cell. Neurosci., 2015, 68. 234-43.

  CaMKII dysregulation is associated with neurological disorders such as but not limited to Angelman syndrome, Prader-Willi syndrome, cerebral ischemia and Alzheimer's disease, and maintenance of the base level of CaMKII autophosphorylation requires T-type calcium channel activity It has been found to be. Blocking the T-type calcium channel may block the binding of calcium to CamKII and reduce inhibitory autophosphorylation, thereby enabling further activation of CamKII and providing an effective therapeutic effect.

  Thus, the present application provides T-type calcium channel antagonists that can inhibit CaMKII autophosphorylation and / or inhibit T-type calcium channels, thereby reducing CaMKII dysregulation such as Angelman syndrome. Useful for the treatment of associated disorders.

I. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

  The terms “for example” and “to” and grammatically equivalent terms thereof are to be interpreted as being followed by the phrase “but not limited” unless specifically stated otherwise.

  The singular form “a” (a / an and the) includes the plural unless specifically stated otherwise.

  The term “about” means “approximately” (eg, plus or minus approximately 10% of the indicated numerical value).

  The term “small molecule” means an organic compound having a molecular weight of about 1000 or less.

  The term “subject” as the subject of treatment means any animal including mammals such as humans.

  The phrase “therapeutically effective amount” refers to an active compound or pharmaceutical agent that elicits a biological or pharmaceutical response that is considered in a tissue, system, animal, individual or human by a researcher, veterinarian, physician or other clinician. Refers to the amount.

  The terms “treating” or “treatment” refer to (1) preventing a disease; eg, having a constitution of a disease, condition or disorder, but have not yet experienced or indicated the pathology or symptoms of the disease Preventing the disease, condition or disorder in an individual; (2) inhibiting the disease; for example, in an individual experiencing or showing a pathology or symptom of the disease, condition or disorder; Inhibiting the disorder (ie, suppressing further progression of pathology and / or symptoms); and (3) improving the disease; eg experiencing or indicating the pathology or symptoms of the disease, condition or disorder Reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease in an individual, Improving the condition or disorder (i.e., to turn around the pathology and / or symptomatology) refers to one or more.

  The term “T-type calcium channel antagonist” refers to the activity of a T-type calcium channel, eg, through binding to the channel, or otherwise inhibiting or blocking its activity, or through reducing the expression of a T-type calcium channel. Refers to the substance to be reduced.

  The phrase “pharmaceutically acceptable” is used herein to refer to reasonable benefits / risk rates without excessive toxicity, irritation, allergic responses or other problems or complications within the scope of sound medical judgment. Is used to refer to compounds, materials, compositions and / or dosage forms suitable for use in contact with human and animal tissues.

  To keep in mind, it will be appreciated that certain features of the invention described in the context of separate embodiments may also be provided in combination in one embodiment. In other words, briefly stated, the various features of the invention described in the context of one embodiment can be provided separately or in any suitable sub-combination.

The following abbreviations and symbols may be used in the present disclosure: CaMKII (calcium / calmodulin-dependent protein kinase II); DNA (deoxyribonucleic acid); dsRNA (double stranded RNA); g (grams); IC ₅₀ (half inhibitory concentration relative to maximum); kg (kilogram); mg (milligram); mRNA (messenger RNA); RNA (ribonucleic acid); RNAi (RNA interference); siRNA (small interfering RNA); wt (weight).

II. Methods of treatment The present invention provides a method of treating a disorder involving CaMKII dysregulation, T-type calcium channel dysregulation, or a combination of CaMKII dysregulation and T-type calcium channel dysregulation in a subject in need thereof. Provided. Subjects can include mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, primates and humans. In some embodiments, the subject is a human. In some embodiments, the treatment comprises reducing or ameliorating neurological symptoms associated with CaMKII dysregulation. In some embodiments, the neurological symptoms are selected from the group consisting of mental retardation, cognitive dysfunction, worsening of long-term potentiation (eg, worsening associated with Angelman syndrome, Prader-Willi syndrome or Alzheimer's disease), and ataxia The In some embodiments, the method employs a therapeutically effective amount of a T-type calcium channel antagonist described herein, or a pharmaceutically acceptable salt thereof, to block inhibitory autophosphorylation of CaMKII. Administration to a subject in need thereof.

  The present disclosure further provides a method of treating a disease selected from Angelman syndrome and Prader-Willi syndrome in a subject in need thereof. In some embodiments, the method comprises administering to a subject in need of treatment a therapeutically effective amount of a T-type calcium channel antagonist described herein. In some embodiments, the disease is Angelman syndrome. In some embodiments, the disease is Prader-Willi syndrome.

  In some embodiments, the treatment comprises reducing or ameliorating neurological symptoms associated with Angelman syndrome or Prader-Willi syndrome. Neurological symptoms can include, but are not limited to, one or more of hyperactivity, abnormal sleep patterns, seizures and ataxia. In some embodiments, the neurological condition includes a seizure. The term “seizure” as used herein includes, but is not limited to absence seizures (eg, typical and atypical atypia), atony seizures, physiological seizures, cluster seizures, incident seizures, Drave syndrome (ie infantile severe myoclonic epilepsy) Or SMEI), focal seizures (ie, partial seizures), focal seizures with secondary generalization, focal seizures with secondary generalized clonic seizures, bald epilepsy, myoclonic seizures, tonic seizures And any one or more of tonic clonic seizures.

  In some embodiments, the subject being treated has symptoms including hyperactivity. In some embodiments, the subject being treated has symptoms that include an abnormal sleep pattern. In some embodiments, the subject being treated has symptoms that include any one or more of the types of seizures defined above. In some embodiments, the subject being treated has symptoms that do not include ataxia. In some embodiments, the subject being treated has symptoms that do not include an abnormal sleep pattern. In some embodiments, the subject to be treated does not include a seizure or is atypical absence seizure, atypical absence, atony seizure, physiological seizure, cluster seizure, incident seizure, Drave syndrome, focal seizure (With or without secondary generalization or secondary generalized clonic seizures), symptoms that do not include one or more of ephemeral epilepsy, myoclonic seizures, tonic seizures or tonic clonic seizures Have.

  Also provided is a method of treating obesity in a subject in need thereof, comprising administering to the subject a T-type calcium channel antagonist. In some embodiments, the treatment includes reducing the weight of the subject. In some embodiments, the treatment includes reducing the amount or rate of weight gain of the subject. In some embodiments, the treatment includes reducing the weight of the subject. In some embodiments, the treatment includes reducing a subject's feeling of hunger or increasing a feeling of fullness.

  Treatment can be carried out by administering a particular compound at an effective dose. Examples of suitable doses in humans include about 1 mg to about 2000 mg, such as about 1 mg to about 2000 mg, about 2 mg to about 2000 mg, about 5 mg to about 2000 mg, about 10 mg to about 2000 mg, about 20 mg to about 2000 mg, about 50 mg. To about 2000 mg, about 100 mg to about 2000 mg, about 150 mg to about 2000 mg, about 200 mg to about 2000 mg, about 250 mg to about 2000 mg, about 300 mg to about 2000 mg, about 400 mg to about 2000 mg, about 500 mg to about 2000 mg, about 1000 mg to about 2000 mg, about 1 mg to about 1000 mg, about 2 mg to about 1000 mg, about 5 mg to about 1000 mg, about 10 mg to about 1000 mg, about 20 mg to about 1000 mg, about 50 mg to about 1 00 mg, about 100 mg to about 1000 mg, about 150 mg to about 1000 mg, about 200 mg to about 1000 mg, about 250 mg to about 1000 mg, about 300 mg to about 1000 mg, about 400 mg to about 1000 mg, about 500 mg to about 1000 mg, about 1 mg to about 500 mg, About 2 mg to about 500 mg, about 5 mg to about 500 mg, about 10 mg to about 500 mg, about 20 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg to about 500 mg, about 1 mg From about 250 mg, from about 2 mg to about 250 mg, from about 5 mg to about 250 mg, from about 10 mg to about 250 mg, from about 20 mg to about 250 mg, from about 50 mg to about 250 mg, from about 100 mg 250 mg, from about 1mg to about 100mg, from about 2mg to about 100mg, from about 5mg to about 100mg, from about 10mg to about 100mg, from about 20mg to about 100mg, include dosages ranging from about 50mg to about 100 mg. The dose can be, for example, about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 20 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 1000 mg, about 1500 mg, or about 1500 mg. It can be 2000 mg. The dose is less than about 2000 mg, less than about 1500 mg, less than about 1000 mg, less than about 5000 mg, less than about 400 mg, less than about 250 mg, less than about 200 mg, less than about 150 mg, less than about 100 mg, less than about 50 mg, less than about 20 mg or about 10 mg It is possible. Each dose can be a dose administered once a day, twice a day, three times a day or four times a day, or in a cycle of less than once a day. Each dose may also be a dose that is administered to an adult equivalent (scale) dose for administration to a pediatric patient.

  Doses are about 100 ng / mL, about 200 ng / mL, 500 ng / mL, about 1 μg / mL, about 2 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 50 μg / mL, about 100 μg / mL. mL, about 200 μg / mL, about 250 μg / mL, or about 500 μg / mL, about 1000 μg / mL, or a range between these numbers, or it is less than these numbers in plasma levels (eg steady state or maximum Level).

  In some embodiments, the treatment is continued for about a week or longer. In some embodiments, the treatment is continued for about 2 weeks or longer. In some embodiments, the treatment is continued for about 3 weeks or longer. In some embodiments, the treatment is continued for about 4 weeks or longer. In some embodiments, the treatment is continued for about 8 weeks or longer. In some embodiments, the treatment is continued for about 12 weeks, or about 13 weeks, or longer. In some embodiments, treatment is continued for about 24 weeks, or about 26 weeks, or longer. In some embodiments, the treatment is continued for about 6 months or longer. In some embodiments, the treatment is continued for about 12 months or longer. In some embodiments, the treatment is continued for about 18 months or longer. In some embodiments, the treatment is continued for about 24 months or longer.

III. T-type calcium channel antagonist The T-type calcium channel antagonist used in any of the methods described herein, or any of its embodiments, can be one or more of the T-type calcium channel antagonists described below.

  A T-type calcium channel antagonist can be an antagonist of a human T-type calcium channel when the subject being treated is a human.

  T-type calcium channel antagonists can be small molecules. Examples of small molecule T-type calcium channel antagonists that can be used in the methods provided herein include, but are not limited to, mibefradil, diltiazem, nifedipine, nitrendipine, nimodipine, nildipine, nigurdipine, nicardipine, nisoldipine, amlodipine, felodipine, Isradipine, Riocidin, Garopamil, Verapamil, Thiapamil, Pimozide, Thioridazine, NNC55-0396, TTL-1177, Anandamide, Benzazepine derivatives, Diphenylbutylpiperidine derivatives (eg Pimozide, Penfluridol, Clopimozide and Flupirylene), Butyrophenone derivatives (eg, Haloperidol) Droperidol, benperidol, triperidol, melperone, lenperone, azaperone and domperi ) And phenylpiperazine derivatives (e.g., anthrafenine, aripiprazole, ciprofloxacin, dapiprazole, dropropidine, etoperidone, itraconazole, ketoconazole, levodolopidine, mepiprazole, naphthopidyl, nefazodone, niaprazine, oxypertin, posaconazole, trazodone, urazodone, And vesnarinone), dihydropyridine derivatives (eg manidipine, nilvadipine, benidipine and efonidipine), flunarizine, anandamide, lomerizine, phenytoin, zonisamide, U-92032, tetralol, tetralol derivatives (eg mibefradil), mibefradil derivatives (eg NNC55-0396 dihydro Chloride), TTA-A2, TTA-A8, TTA- 1,4-aminomethyl-4-fluoropiperidine (TTA-P2), TTA-Q3, TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944, derivatives of succinimide antispasmodic agents (eg ethosuximide, fence) Mesuximide, also known as succinimide and methosuccimide, N-desmethylmethosuccimide, also known as (α) -methyl- (α) -phenyl-succinimide), and efonidipine (eg (R) -efonidipine) , Trimetadione, dimetadione, ABT-639, TTL-1177, KYSO5044, and curtoxin. Any of the T-type calcium channel inhibitors can be in the form of a pharmaceutically acceptable salt. The structure of a specific T-type calcium channel inhibitor is shown below:

  In some embodiments, T-type calcium channel small molecule modulators can be found in Giordanetto et al, “T-type calcium channels inhibitors: a patent review,” Expert Opin. Ther. Pat., 2011, 21, 85-101. Patent specifications and patent application publications listed, the entire contents of each of which are listed by reference, such as WO 2004/035000 pamphlet, WO 93/04047 pamphlet, WO 2006/098969 pamphlet. International Publication No. 2009/009015, International Publication No. 2007/002361, International Publication No. 2007/002884, International Publication No. 2007/120729, International Publication No. 2009/054982 Pamphlet, International Publication No. 2009. / 054983 pamphlet, International Publication No. 2009/054984 pamphlet, US Patent Application Publication No. 2009/0270413, International Publication No. 2008/110008, International Publication No. 2009/146539, International Publication No. 2009/146540, US Pat. No. 8,133,998 Description, WO 2010/083264 pamphlet, WO 2006/023881 pamphlet, WO 2006/023883 pamphlet, WO 2005/007124 pamphlet, WO 2005/009392 pamphlet, US patent Application Publication No. 2005/245535, International Publication No. 2007/073497, International Publication No. 2007/07852, International Publication No. 20 8/033447 pamphlet, International Publication No. 2008/033456 pamphlet, International Publication No. 2008/033460 pamphlet, International Publication No. 2008/033464 pamphlet, International Publication No. 2008/033465 pamphlet, International Publication No. 2008/050200 pamphlet. International Publication No. 2008/117148, International Publication No. 2009/056934, European Patent No. 1568695, International Publication No. 2008/007835, Korean Patent No. 754325, US Pat. No. 7319098 US Patent Application Publication No. 2010/0004286, European Patent No. 1757590, Korean Patent Application Publication No. 2009/044924, US Patent Application No. 2010/094006, International Publication No. 2009/035307, US Patent Application Publication No. 2009/0325979, Korean Patent No. 75758317, International Publication No. 2008/018655, US Patent Application It may be selected from the group consisting of those described in Japanese Patent Application Publication No. 2008/0293786 and US Patent Application Publication No. 2010/0056545.

  In some embodiments, the T-type calcium channel antagonist is a small molecule. In some embodiments, the small molecule has a molecular weight of 1000 or less, such as about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400. Or a molecular weight of less than or in the range of about 100 to about 500, about 200 to about 500, about 200 to about 400, about 300 to about 400, or about 300 to about 500.

In some embodiments, the T-type calcium channel antagonist is a selective T-type calcium channel antagonist. “Selective” in this context means that the T-type calcium channel antagonist is another type of calcium, such as any one or more of L-type, N-type, P-type, Q-type and / or R-type calcium channels. It means antagonizing the T-type calcium channel more strongly than the channel, for example L-type calcium channel. Selectivity can be measured, for example, by comparing the IC ₅₀ of a compound in inhibiting a T-type calcium channel with the IC ₅₀ in inhibiting other types of calcium channels, for inhibiting a T-type channel. If IC ₅₀ of less than IC ₅₀ of to inhibit other types of calcium channels, compounds are considered to be selective. An IC ₅₀ ratio of 0.1 (or less) means 10 times (or more) selectivity. An IC ₅₀ ratio of 0.01 (or less) means 100 times (or more) selectivity. An IC ₅₀ ratio of 0.001 (or less) means 1000 times (or more) selectivity. In some embodiments, the T-type calcium channel antagonist is other to the T-type calcium channel, such as any one or more of L-type, N-type, P-type, Q-type and / or R-type calcium channels. 10 times or more, 100 times or more or 1000 times or more selectivity compared to other types of calcium channels, such as L-type calcium channels.

  In some embodiments, the T-type calcium channel antagonist is fensuximide, methosuccimide, methyl l-phenyl-succinimide, the R isomer of efonidipine, trimetadione, dimetadione, mibefradil, TTA-A2, TTA-A8, TTA-P1. , TTA-P2, TTA-Q3, TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944, ABT-639, TTL-1177, KYSO5044, NC55-0396 dihydrochloride, Kurtoxin or its derivatives A selective T-type calcium channel inhibitor selected from the group consisting of:

  In some embodiments, the T-type calcium channel antagonist is selected from the group consisting of mibefradil, efonidipine, TTL-1177, nickel, and combinations thereof.

  In some embodiments, the T-type calcium channel antagonist is mibefradil.

  In some embodiments, the T-type calcium channel antagonist is MK-5395.

  In some embodiments, the T-type calcium channel antagonist is MK-6526.

  In some embodiments, the T-type calcium channel antagonist is MK-8998.

  In some embodiments, the T-type calcium channel antagonist is Z944.

  In some embodiments, the T-type calcium channel antagonist can be other than ethosuximide.

  In some embodiments, a T-type calcium channel antagonist can be a molecule that does not act as a pore blocker for T-type calcium channels. The T-type calcium channel antagonist can be, for example, an allosteric inhibitor of T-type calcium channel.

In some embodiments, the T-type calcium channel antagonist is Na _V 1.1, Na _V 1.2, Na _V 1.3, Na _V 1.4, Na _V 1.5, Na _V 1.6, One such as a sodium channel with Na _V 1.7, Na _V 1.8 or Na _V 1.9α subunit and / or Na _V β1, Na _V β2, Na _V β3, Na _V β4 subunit, or It may be one that does not substantially affect multiple sodium channels. T-type calcium channel antagonists may be selective for T-type calcium channels compared to inhibition of sodium channels, for example, at least 2-fold, at least 5-fold, at least 10 (for example, expressed in terms of K _i ). Have a selectivity of at least 20 times, at least 100 times, at least 500 times or at least 1000 times. T-type calcium channel inhibitors do not substantially reduce non-inactivated sodium currents in thalamic cortical neurons, eg, about 20% or less, about 10% or less, about 5% or less, about It may be one that reduces the inactivated sodium current by 2% or less or about 1% or less.

In some embodiments, the T-type calcium channel antagonist is a calcium-activated potassium channel (BK channel, SK channel, IK channel), an inward rectifier potassium channel (ROMK, GPCR-controlled, ATP-sensitive), serial pore Domain potassium channels (TWIK (TWIK-1, TWIK-2, KCNK7), TREK (TREK-1, TREK-2, TRAAK), TASK (TASK-1, TASK-3, TASK-5), TALK (TASK-2) , TALK-1, TALK-2), THIK (THIK-1, THIK-2), TRESK), or one or more potassium channels such as voltage-gated potassium channels (hERG, KvLQT) It may not be. T-type calcium channel antagonists may be selective for T-type calcium channels compared to inhibition of potassium channels, for example, at least 2-fold, at least 5-fold, at least 10 (eg when expressed in terms of K _i ). Have a selectivity of at least 20 times, at least 100 times, at least 500 times or at least 1000 times.

In some embodiments, the T-type calcium channel antagonist substantially affects one or more GABA receptors, such as GABA _A receptors, GABA _A- ρ subclass (GABA _C ) receptors or GABA _B receptors. It may be a thing which does not exert. In some embodiments, the T-type calcium channel antagonist is, for example, an α-subunit (GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6), β-subunit (GABRB1, GABRB2, GABRB3), γ-subunit ( GABRG1, GABRG2, GABRG3), δ-subunit (GABRD), ε-subunit (GABRE), π-subunit (GABRP), θ-subunit (GABRQ), particularly GABA _A receptors such as GABARA5, GABRB3 and GABRG5 It may be one that does not substantially affect one or more subunits of the body. T-type calcium channel antagonists, as compared to the inhibition of GABA receptors, e are selective for T-type calcium channels, for example, (e.g., when expressed in terms of the K _i or binding affinity) of at least 2-fold, at least Selectivity of 5 times, at least 10 times, at least 20 times, at least 100 times, at least 500 times or at least 1000 times.

  In some embodiments, the T-type calcium channel antagonist may not cause one or more of the following side effects or adverse events after administration to an animal such as a human: liver injury, animal liver morphology Changes, functional changes in the liver of the animals, kidney damage, morphological changes in the kidneys of the animals, functional changes in the kidneys of the animals, systemic lupus erythematosus, suicide intentions, suicidal behavior, suicidal ideation, increased suicide risk, development of depression Or worsening, abnormal emotional or behavioral changes, birth defects, allergic reactions.

  In some embodiments, a T-type calcium channel antagonist may not cause one or more of the following side effects or adverse events after administration to an animal: gastrointestinal adverse events (eg, eating disorders, Vague abnormalities, nausea and vomiting, convulsions, upper and abdominal pain, weight loss, diarrhea, gingival enlargement and tongue enlargement); hematopoietic adverse events such as leukopenia, agranulocytosis, pancytopenia ( Adverse events in the nervous system (eg, drowsiness, headache, dizziness, euphoria, including myelosuppression and eosinophilia); neurological responses, sensory responses, or psychiatric or psychogenic modulation Sensation, hiccups, irritability, hyperactivity, lethargy, fatigue, ataxia, confusion, sleep disturbance, night surprises, lack of concentration, aggression, paranoid psychosis, increased libido, or worsening with obvious suicide intentions Depressive state); ectodermal adverse events including dermatopathological signs (eg urticaria, Stevens-Johnson syndrome, systemic lupus erythematosus, pruritic rash and hirsutism); special sensory organs such as myopia Adverse events; and genitourinary events such as genital bleeding or microscopic hematuria.

  In some embodiments, the T-type calcium channel antagonist is an antibody. Various methods for the preparation of antibodies are known in the art. Antibodies: See A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989). For example, antibodies can be prepared by immunizing a suitable mammalian host with a sample containing whole cells isolated from a patient. The antibody may be transfected with an antibody gene into a suitable bacterial cell or mammalian cell host by cell culture techniques including the production of monoclonal antibodies as described herein or to allow the production of recombinant antibodies. Can be manufactured.

  In some embodiments, the antibody is a monoclonal antibody. A “monoclonal antibody” is an antibody obtained from a substantially homogeneous population of antibodies, ie, the antibodies that make up the population are identical except for naturally occurring mutations that may be present in small amounts.

  In some embodiments, the antibodies provided herein can be produced by recombinant methods. In some embodiments, the antibody is a “humanized” or human antibody. “Humanized” or human antibodies can also be prepared and are preferably used in therapeutic situations. Methods for humanizing mouse and other non-human antibodies are made by substituting one or more of the non-human antibody sequences with the corresponding human antibody sequences, and are well known. For example, Jones et al., Nature, 1986, 321, 522-25; Riechmann et al., Nature, 1988, 332, 323-27; Verhoeyen et al., Science, 1988, 239, 1534-36, Carter et al ., Proc. Natl. Acad. Sci. USA, 1993, 89, 4285; and Sims et al., J. Immunol., 1993, 151, 2296. These humanized antibodies are designed to minimize undesirable immunological responses to rodent anti-human antibody molecules, thereby reducing the duration and effect of therapeutic application of those portions to human recipients. Limited. Accordingly, preferred antibodies for use in the treatment methods described herein are fully human antibodies or humanized antibodies that have high affinity but low or no antigenicity in the subject.

  In some embodiments, the T-type calcium channel antagonist is an oligonucleotide inhibitor. Examples of oligonucleotide inhibitors include, but are not limited to, antisense oligonucleotides, RNAi, dsRNA, siRNA and ribozymes. In some embodiments, the T-type calcium channel antagonist is an siRNA. As used herein, "antisense oligonucleotide" refers to a single-stranded DNA or RNA extension, usually chemically modified, whose sequence (3'-5 ') is the sense of the mRNA molecule. It is complementary to the sequence. Antisense molecules effectively inhibit gene expression by forming RNA / DNA duplexes. Antisense is understood to be the action by various mechanisms including physically blocking the ability of ribosomes to move along messenger RNA and increasing the rate at which mRNA is degraded in the cytosol. ing.

  To avoid degradation by DNAse, antisense oligonucleotides can be chemically modified. For example, phosphorothioate oligodeoxynucleotides are stabilized by substituting one of the non-bridged phosphoryl oxygens of the DNA with a sulfur moiety, preventing degradation by nucleases. The increased stability of antisense oligonucleotides has been substituted with 2-methoxyethyl (MOE), as generally described in US Pat. No. 6,419,991 and US Publication No. 2003/0158143, which are incorporated by reference. It can also be carried out using a molecule having a skeleton. Thus, antisense oligonucleotides can be modified to enhance in vivo stability compared to unmodified oligonucleotides of the same sequence. The modification may be, for example, a (2'-O-2-methoxyethyl) modification. The oligonucleotide may have a phosphorothioate backbone overall, the sugar moieties of nucleotides 1-4 and 18-21 may have 2′-O-methoxyethyl modifications, and other nucleotides may be 2 It may be a '-deoxynucleotide.

  It is understood in the art that an antisense oligonucleotide need not have 100% identity with the complementary sequence of its target sequence in order to be effective. The antisense oligonucleotide can thus have a sequence that is at least about 70% identical to the complementary sequence of the target sequence. In one embodiment, the antisense oligonucleotide can have a sequence that is at least about 80% identical to the complementary sequence of the target sequence. In other embodiments, they have a sequence that is at least about 90% identical or at least about 95% identical to the complementary sequence of the target sequence, taking into account some base gaps or mismatches. Identity can be determined, for example, using the BLASTN program, which is software of the Computer Group (GCG) at the University of Wisconsin.

  Antisense oligonucleotides according to the present invention are typically between 7 and 100 nucleotides in length. In one embodiment, the antisense oligonucleotide comprises about 7 to about 50 nucleotides or nucleotide analogues. In another embodiment, the antisense oligonucleotide comprises from about 7 to about 35 nucleotides or nucleotide analogues. In other embodiments, the antisense oligonucleotide comprises from about 12 to about 35 nucleotides or nucleotide analogs, and from about 15 to about 25 nucleotides or nucleotide analogs.

  The oligonucleotide inhibitor according to the present invention may be a siRNA molecule targeting a gene of interest whose sequence corresponds to a part of the gene. The RNA molecules used in the present invention generally comprise an RNA portion and some further portion (eg, deoxyribonucleotide portion).

  The present disclosure further encompasses ribozyme oligonucleotide modulators that specifically target mRNA encoding a protein of interest, such as a protein containing a T-type calcium channel. A ribozyme is an RNA molecule with enzymatic activity that allows the ribozyme to cleave other discrete RNA molecules repeatedly in a nucleotide sequence specific manner. Such enzymatic RNA molecules can target virtually any mRNA transcript and effective cleavage can be performed in vitro. Kim et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 8788; Haseloff et al., Nature, 1988, 334, 585; Cech, JAMA, 1988, 260, 3030; and Jefferies et al., Nucleic Acids Res., 1989, 17, 1371.

  Typically, a ribozyme comprises two parts that are held in close proximity, an mRNA binding part having a sequence complementary to the target mRNA sequence and a catalytic part that acts to cleave the target mRNA. The ribozyme first has an action of recognizing and binding to the target mRNA by complementary base pairing through the binding portion of the ribozyme with the target mRNA. After specifically binding to its target, the ribozyme catalyzes the cleavage of the target mRNA. Such strategic cleavage abolishes the ability of the target mRNA to direct the synthesis of the encoded protein. After binding to and cleaving the mRNA target, the ribozyme is released and can repeatedly bind to and cleave with a new target mRNA molecule.

  In some embodiments, the selective T-type calcium channel antagonist substantially crosses the blood brain barrier.

  In some embodiments, the selective T-type calcium channel antagonist does not substantially cross the blood brain barrier.

  In some embodiments, the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels. In some embodiments, the T-type calcium channel antagonist is a small molecule described herein.

In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.1. In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.2. In some embodiments, the T-type calcium channel antagonist selectively targets Ca _V 3.3. “Selective” in this context means that a T-type calcium channel antagonist antagonizes one type of T-type calcium channel more strongly than another type of calcium channel, eg, Ca _V 3.2 or Ca _{V V} 3.3 or strongly antagonizes _Ca V 3.1 than _both, Ca V 3.1 or _Ca V 3.3 or strongly antagonizes _Ca V 3.2 than both, _Ca V 3 .1 or Ca _V 3.2 or both means stronger antagonizing of Ca _V 3.3. Selectivity can be measured, for example, by comparing the IC ₅₀ of a compound in inhibiting one type of T-type calcium channel with the IC ₅₀ in inhibiting other types of T-type calcium channels. If IC ₅₀ of for inhibiting T-type channel is lower than the IC ₅₀ of to inhibit other types of T-type calcium channel, the compounds are considered to be selective. An IC ₅₀ ratio of 0.1 (or less) means 10 times (or more) selectivity. An IC ₅₀ ratio of 0.01 (or less) means 100 times (or more) selectivity. An IC ₅₀ ratio of 0.001 (or less) means 1000 times (or more) selectivity. In some embodiments, the selectivity for Ca _V 3.1, Ca _V 3.2 or Ca _V 3.3 is 10 times or more, 100 times or more or 1000 times or more.

In some embodiments, the T-type calcium channel antagonist is, for example, Na _V 1.1, Na _V 1.2, Na _V 1.3, Na _V 1.4, Na _V 1.5, Na _V 1.6. , Na _V 1.7, Na _V 1.8 or Na _V 1.9α subunits and / or sodium channels such as sodium channels with Na _V β1, Na _V β2, Na _V β3, Na _V β4 subunits Also selectively target T-type calcium channels (eg, Ca _V 3.1, Ca _V 3.2 and / or Ca _V 3.3). T-type calcium channel antagonists may be selective for T-type calcium channels compared to inhibition of sodium channels. Selectivity can be determined, for example, by comparing the IC ₅₀ of a compound in inhibiting one or more types of T-type calcium channels to its IC ₅₀ in inhibiting one or more types of sodium channels. A compound is considered selective if it can be measured and the IC _{50 in} inhibiting the T-type calcium channel is lower than the IC _{50 in} inhibiting the sodium channel. An IC ₅₀ ratio of 0.1 (or less) means 10 times (or more) selectivity. An IC ₅₀ ratio of 0.01 (or less) means 100 times (or more) selectivity. An IC ₅₀ ratio of 0.001 (or less) means 1000 times (or more) selectivity. In some embodiments, the selectivity for T-type calcium channels is 10 or more, 100 or more, or 1000 or more.

  The effect of a compound that inhibits a T-type calcium channel can vary depending on the state of the T-type calcium channel that the T-type calcium channel antagonist inhibits. T-type calcium channels may show different states depending on the cell membrane potential. T-type calcium channel antagonists that are effective in the methods described herein block T-type calcium channels when the membrane potential is in the range of about −60 mV to about −30 mV, such as preferably about −40 mV. T-type calcium channel antagonists can be included. A membrane potential in the range of “about −60 to about −30 mV” can include a membrane potential in the range of −70 mV to −20 mV, or in the range of −65 mV to −25 mV, and for example, about −40 mV to about −30 mV. About −50 mV to about −30 mV, about −70 mV to about −30 mV, about −50 mV to about −40 mV, about −60 mV to about −40 mV, about −70 mV to about −40 mV, about −60 mV to about −50 mV, and A range of membrane potentials from about −70 to about −50 mV, and about −30 mV, about −40 mV, about −50 mV, and about −60 mV can also be included. In some embodiments, T-type calcium channel antagonists that are effective in the methods described herein include membrane potentials in the range of about −100 mV to about −80 mV, such as preferably about −90 mV. T-type calcium channel antagonists that block T-type calcium channels can be included. A membrane potential in the range of “about −100 to about −80 mV” can include a membrane potential in the range of −110 mV to −70 mV, or in the range of −105 mV to −75 mV, and also includes, for example, about −100 mV to about − A range of membrane potentials of 80 mV, about −90 mV to about −80 mV and about −100 mV to about −90 mV, and about −100 mV, about −90 mV and about −80 mV can also be included.

  Without being bound by any theory, T-type calcium channel antagonists that are effective in the methods described herein include T-type when the membrane potential is in the range of about −60 mV to about −30 mV, such as −40 mV. A T-type calcium channel antagonist may be included that selectively blocks calcium channels relative to blocking T-type calcium channels when the membrane potential is in the range of about −100 mV to about −80 mV, eg, about −90 mV. Conceivable.

An effective T-type channel inhibitor is an IC ₅₀ for inhibiting a T-type calcium channel when the membrane potential is about −40 mV, ie about 10 μM or less, for example about 1 μM or less, about T-type calcium channels can be inhibited at 500 nM or less, about 100 nM or less, about 50 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less. An effective T-type calcium channel antagonist can selectively inhibit a T-type calcium channel at a membrane potential of about -40 mV compared to inhibition of the T-type calcium channel at a membrane potential of about -90 mV. For example, the ratio of the IC ₅₀ of a T-type calcium channel antagonist that selectively inhibits T-type calcium channels at a membrane potential of about −40 mV to that upon inhibition of T-type calcium channels at a membrane potential of about −90 mV is about 1: 2 or less, such as about 1: 5 or less, about 1:10 or less, about 1:20 or less, about 1:50 or less, about 1: 100 or less, about 1 : 500 or less, about 1: 1000 or less. In some embodiments, the selectivity for inhibiting about −40 mV T-type calcium channel is 2 × or more, 5 × or more, 10 × compared to inhibiting about −90 mV T-type calcium channel. Or more than, 100 times or more, or 1000 times or more.

T-type channel inhibitors that are effective have an IC ₅₀ for inhibiting T-type calcium channels when the membrane potential is about −90 mV, ie about 10 μM or less, such as about 1 μM or less, about T-type calcium channels can be inhibited at 500 nM or less, about 100 nM or less, about 50 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less. An effective T-type calcium channel antagonist can selectively inhibit a T-type calcium channel at a membrane potential of about -90 mV compared to inhibition of the T-type calcium channel at a membrane potential of about -40 mV. For example, the ratio of the IC ₅₀ of a T-type calcium channel antagonist that selectively inhibits T-type calcium channels at a membrane potential of about −90 mV to that upon inhibition of T-type calcium channels at a membrane potential of about −40 mV is about 1: 2 or less, such as about 1: 5 or less, about 1:10 or less, about 1:20 or less, about 1:50 or less, about 1: 100 or less, about 1 : 500 or less, about 1: 1000 or less. In some embodiments, the selectivity for inhibiting about −90 mV T-type calcium channel is 2 × or more, 5 × or more, 10 × compared to inhibiting about −40 mV T-type calcium channel. Or more than, 100 times or more, or 1000 times or more.

  All compounds and their pharmaceutically acceptable salts can exist with other substances such as water and solvents (eg hydrates and solvates) or can be isolated. In some embodiments, a compound provided herein or a pharmaceutically acceptable salt thereof is substantially isolated. “Substantially isolated” means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. As a substantial separation, the compound or salt thereof provided herein is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95 on a weight basis. %, At least about 97%, or at least about 99%. Methods for isolating compounds and their salts are routine in the art.

  As used herein, “pharmaceutically acceptable salt” refers to a derivative of a disclosed compound that has been modified by converting an existing acid or base moiety in the parent compound to its salt form. Point to. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts of the present application include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Usually, such salts are the free acid or base form of these compounds and their stoichiometric amount of the appropriate base or acid in water, or an organic solvent, or a mixture of the two. In general, a non-aqueous medium such as ether, ethyl acetate, alcohol (for example, methanol, ethanol, isopropanol or butanol) or acetonitrile is preferable. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

IV. Combination Therapy One or more additional therapeutic agents can be used in combination with the compounds provided herein for the treatment of Angelman syndrome or Prader-Willi syndrome. Examples of additional therapeutic agents include, but are not limited to, calcium channel antagonists (including L and T type antagonists), anticonvulsants, GABA (A) receptor agonists and positive allosteric modulators, or gene therapy or gene reactivation. Activation therapy (Bailus et al., The prospect of molecular therapy for Angelman syndrome and other monogenic neurologic disorders, BMC Neuroscience, 2014, 15, 76).

  In some embodiments, treatment with a T-type calcium channel antagonist can also be provided in the absence of additional drugs to treat Angelman syndrome or Prader-Willi syndrome. In some embodiments, treatment can be performed with one T-type calcium channel antagonist. In some embodiments, treatment with a T-type calcium channel antagonist can be provided in the absence of an anticonvulsant.

  One or more additional medicinal products can be administered to the patient simultaneously or sequentially using the same or different dosing schedules, as determined by the particular combination used and the judgment of the prescribing physician Is done.

  Examples of calcium channel antagonists include, but are not limited to, T-type calcium channel antagonists and L-type calcium channel antagonists described herein. In some embodiments, the additional calcium channel antagonist is selected from T-type calcium channel antagonists provided herein. In some embodiments, the additional calcium channel antagonist is an L-type calcium channel antagonist. In some embodiments, the additional calcium channel antagonist is a T-type calcium channel antagonist. In some embodiments, the additional calcium channel antagonist is a T-type calcium channel antagonist selected from the group consisting of mibefradil, MK-5395, MK-6526, MK-8998, and Z944. In some embodiments, additional calcium channel antagonists are T-type calcium channel antagonists and L-type calcium channel antagonists. In some embodiments, the additional calcium channel antagonist is a T-type calcium channel antagonist or an L-type calcium channel antagonist selected from the group consisting of ACT-28077, mibefradil and TTL-1177. In some embodiments, the additional calcium channel antagonist is mibefradil.

  Examples of anticonvulsants include, but are not limited to, acetazolamide, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, gabapentin, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel , Piracetam, phenobarbital, phenytoin, pregabalin, primidone, retigabine, rufinamide, valproate (eg sodium valproate), styripentol, tiagabine, topiramate, vigabatrin and zonisamide.

  Examples of GABA (A) receptor agonists include gaboxadol, bamalzole, γ-aminobutyric acid, gabamide, γ-amino-β-hydroxybutyric acid, gaboxadol, ibotenic acid, isoguvacine, isonipecotic acid , Muscimol, phenibut, picamilon, progabide, quisqualamine, SL75102 and thiomucimol.

  Examples of positive allosteric modulators of GABA (A) receptors include evamectin (eg, ivermectin), barbiturates (eg, phenobarbital), benzodiazepines (eg, adinazolam, alprazolam, bentazepam, bretazenil, bromazepam, brotizolam, camazepam, chlordiazepoxide, Synazepam (cinazepam), sinorazepam, clobazam, clonazepam, clonazolam, chlorazepate, clothiazepam, cloxazolam, delorazepam, diazepam, diclazepam (diclazepam), estazolam, ethyl fluzepamazom, fluzepamazom (Flubromazolam), flunitrazepam (flunitrazepam), flurazepam (flurazepam), flutazo , Frutoprazepam, Harazepam, Ketazolam, Roprazolam, Lorazepam, Lormetazepam (lormetazepam), Medazepam, Mexazolam, Midazolam, Nifoxipam, Nimetazepam (Nimetazepam), Nitrazepam, Pamzepamazem , Pyrazolam, quazepam, rilmazafone, temazepam, thienalprazolam, tetrazepam and triazolam), bromides (eg potassium bromide, carbamates (eg meprobamate, carisoprodol)), chloralose , Chlormezanone, clomethiazole, dihydroergoline (eg ergoloid (dihydroergotoxin)), Zepin, ethifoxine, imidazole (eg etomidate), kava lactone (present in kava), lorecresol, neurostimulatory steroids (eg allopregnanolone, ganazolone), non-benzodiazepines (eg zaleplon, zolpidem, zopiclone, eszopiclone), petrichloral (Petrichloral), phenols (eg propofol), piperidinediones (eg glutethimide, metiprilone), propanidid, pyrazolopyridine (eg etazolate), quinazolinones (eg metaqualone), calvarial components, styripentol, sulfonylalkanes (eg sulfone) Methane, tetronal, trional), and valerian components (eg, valeric acid, valerenic acid).

  In some embodiments, the therapy can be performed as a monotherapy. In some embodiments, the therapy can be performed in the absence of additional antispasmodic therapy. The therapy can be performed in the absence of any of the additional agents described in this section. For example, therapies include acetazolamide, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, gabapentin, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perranpanel, piracetam, phenobarbital, phenytoin, pregabine This can be done in the absence of additional antispasmodic agents such as primidone, retigabine, rufinamide, valproate (eg sodium valproate), stilipentol, tiagabine, topiramate, vigabatrin or zonisamide.

  Gene reactivation therapy involves the administration of a compound that activates a paternal (or maternal) copy of UBE3A. Examples include topoisomerase inhibitors (including topoisomerase I and II inhibitors) such as topotecan, irinotecan, etoposide and dexrazoxane, and Huang et al., "Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons", Nature, 2011 , 481, 185-89.

  In some embodiments, one or more additional therapies that can be used in combination with the T-type calcium channel antagonists provided herein include, but are not limited to, physical therapy, occupational therapy, communication therapy and behavioral therapy. Is mentioned. In some embodiments, the one or more additional therapeutic agents and one or more additional therapies that can be used in combination with the T-type calcium channel antagonists provided herein include physical therapy, occupational therapy , Selected from the group consisting of communication therapy and behavioral therapy.

V. Pharmaceutical Composition The T-type calcium channel inhibitor used in the methods described herein can be administered in the form of a pharmaceutical composition. Thus, the disclosure of the present invention includes a T-type calcium channel inhibitor and at least one pharmaceutically used in the manufacture of a medicament for the treatment of a claimed method or a condition described herein. And an acceptable carrier. These compositions can be prepared by methods known in the pharmaceutical art and can be administered by various routes. Administration includes topical administration (including transdermal, epidermal, intraocular, and transmucosal, including intranasal, intravaginal and rectal delivery), pulmonary administration (eg, inhalation or infusion of powder or aerosol, such as by nebulizer, Intraductal or intranasal), oral or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or infusion or infusion, or intracranial (eg, subarachnoid or intraventricular) administration. Parenteral administration may be in the form of a single bolus administration or by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners and the like may be necessary or desirable.

  This pharmaceutical composition is a combination of one or more pharmaceutically acceptable carriers (excipients) and as a T-type calcium channel inhibitor (pharmaceutically acceptable salt form) as an active ingredient. Good). In some embodiments, the composition is suitable for topical administration. In preparing the compositions of the present invention, the active ingredient is typically mixed with or diluted with an excipient, or in the form of a capsule, sachet, paper or other container, etc. In such a carrier. When an excipient is used as a diluent, it may be a solid, semi-solid or liquid material that functions as a vehicle, carrier or medium for the active ingredient. Thus, the composition can be a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (in solid or liquid medium), eg up to 10% by weight of active compound Ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

  In preparing the formulation, the T-type calcium channel inhibitor can be milled to an appropriate particle size prior to combining with other ingredients. If the active compound is substantially insoluble, it can be ground to a particle size of less than 200 mesh. When the active compound is substantially water soluble, the particle size can be adjusted by milling to provide substantially the same distribution (eg, about 40 mesh) in the formulation.

  The compounds of the present invention may be milled using known milling processes such as wet milling in order to obtain a particle size suitable for tablet formation and other dosage forms. Finely divided (nanoparticulated) preparations of the compounds of the invention can be prepared by processes known in the art.

  Examples of some suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gum tragacanth, gelatin, calcium silicate, crystalline cellulose, polyvinylpyrrolidone, cellulose, water , Syrup and methylcellulose. The formulation further includes lubricants (eg, talc, magnesium stearate and mineral oil), wetting agents, emulsifiers and suspensions, preservatives (eg, methyl and propyl esters of hydroxybenzoic acid), sweeteners, and flavors. Can do.

  In some embodiments, the pharmaceutical composition comprises silicified crystalline cellulose (SMCC) and at least one compound described herein or a pharmaceutically acceptable salt thereof. In some embodiments, the silicified crystalline cellulose comprises about 98% crystalline cellulose and about 2% silicon dioxide on a weight basis.

  In some embodiments, a wet granulation process is used to produce the composition. In some embodiments, a dry granulation process is used to make the composition.

  The composition can be formulated in unit dosage form, with each dosage unit containing from about 5 to about 1,000 mg (1 g), more usually from about 100 mg to about 500 mg of active ingredient. In some embodiments, each dosage unit contains about 10 mg of active ingredient. In some embodiments, each dosage unit contains about 50 mg of active ingredient. In some embodiments, each dosage unit contains about 25 mg of active ingredient. The term “unit dosage form” refers to physically divided units suitable for single administration for human subjects and other mammals, each unit in combination with a suitable pharmaceutical excipient, Contains a predetermined amount of active substance calculated to obtain the desired therapeutic effect.

  The ingredients used to formulate the pharmaceutical composition are of high purity and substantially free of potentially harmful contaminants (eg, at least domestic food grade, generally at least analytical grade, more typically Is at least pharmaceutical grade). Particularly when ingested by humans, the composition is preferably manufactured or formulated in accordance with pharmaceutical manufacturing quality control standards as defined in relevant regulations of the US Food and Drug Administration. For example, suitable formulations may be sterile and / or substantially isotonic and / or fully compliant with all US Food and Drug Administration quality control standards.

  The active compound can be effective over a wide dosage range but is usually administered in a therapeutically effective amount. However, the amount of compound actually administered will depend on the condition being treated, the route of administration chosen, the actual compound being administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. It will be understood that this will be determined by the physician depending on the situation.

  The therapeutic dosage of the compounds of the present invention can vary depending on, for example, the particular use in which the treatment is being made, the manner of administration of the compound, the condition of the patient and the judgment of the prescribing physician. The ratio or concentration of a compound of the invention in a pharmaceutical composition can vary depending on many factors such as dosage, chemical properties (eg, hydrophobicity) and route of administration. For example, the compounds of the invention can be provided as a physiological buffered aqueous solution containing about 0.1 to about 10% (w / v) of the compound for parenteral administration. Some typical dose ranges are from about 1 μg / kg to about 1 g / kg body weight per day. In some embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg body weight per day. The dosage will depend on such variables, the severity of the disease, the overall health status of the particular patient, the relative biological effectiveness of the selected compound, the composition of the excipients and the route of administration. Has a tendency. Effective doses can be extrapolated from dose-response curves derived from model test systems in vitro or in animals. Effective doses for humans are, for example, about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, It can be 95 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1000 mg. The dose can be administered, for example, once a day, twice a day, three times a day, or four times a day.

  In some embodiments, when the T-type calcium channel antagonist is mibefradil, mibefradil can be administered at a dose of, for example, about 0.1 mg, 0.3 mg, 1 mg, 3 mg, 5 mg, 15 mg, 10 mg or 30 mg. The dose can be administered, for example, once a day, twice a day, three times a day, or four times a day.

  In some embodiments, when the T-type calcium channel antagonist is MK-5395, the MK-5395 is, for example, about 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 30 mg / kg. It can be administered at a dose of kg or 100 mg / kg. The dose can be administered, for example, once a day, twice a day, three times a day, or four times a day.

  In some embodiments, when the T-type calcium channel antagonist is MK-6526, the MK-6526 is, for example, about 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 30 mg / kg. It can be administered at a dose of kg or 100 mg / kg. The dose can be administered, for example, once a day, twice a day, three times a day, or four times a day.

  In some embodiments, when the T-type calcium channel antagonist is MK-8998, the MK-8998 is, for example, about 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 30 mg / kg. It can be administered at a dose of kg or 100 mg / kg. The dose can be administered, for example, once a day, twice a day, three times a day, or four times a day.

  In some embodiments, when the T-type calcium channel antagonist is Z944, the Z944 is, for example, about 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 30 mg / kg or 100 mg / kg. Can be administered at a dose of kg. The dose can be administered, for example, once a day, twice a day, three times a day, or four times a day.

  When preparing solid compositions such as tablets, the principal active ingredient is mixed with pharmaceutical excipients to form a solid preformulation composition containing a homogeneous mixture of the compounds of the invention. Uniformity of these pre-formulated compositions typically means that the active ingredient is uniformly dispersed throughout the composition, such that the composition is an equally effective unit dosage form (eg, tablets, pills and capsules). ) Refers to the state that can be easily subdivided. This solid preformulation is then subdivided into unit dosage forms of the type described above containing, for example, from about 0.1 to about 1000 mg of the active ingredient of the present invention.

  Solution forms in which the compounds and compositions of the invention can be formulated for oral administration or infusion include aqueous solutions, appropriately flavored syrups, suspensions in water or oil, and cottonseed oil, sesame oil, coconut oil, or peanut oil Edible oil-flavored emulsions, and elixirs and similar pharmaceutical vehicles.

  Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered by the oral or nasal respiratory route to obtain a local or systemic effect. The composition can be atomized by use of an inert gas. Nebulized solution may breathe directly from the nebulizer or the nebulizer can be attached to a mask, tent or intermittent positive pressure breathing device. Solutions, suspensions or powdered compositions may be administered in an appropriate manner, orally or intranasally from a device that delivers the formulation.

  A topical formulation can contain one or more carriers. In some embodiments, the ointment may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petrolatum, and the like. The creamy carrier composition can be a water-based composition in combination with glycerol and one or more other ingredients (eg, glycerin monostearate, PEG-glycerin monostearate and cetyl stearyl alcohol). The gel can be formulated by using isopropyl alcohol and water and appropriately combining with other components (for example, glycerol, hydroxyethyl cellulose, etc.). In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5% by weight of a compound of the invention. .

  The amount of the compound or composition administered to a patient varies depending on the substance to be administered, the purpose of administration such as prevention or therapy, the condition of the patient, the administration method, and the like. For therapeutic use, the composition can be administered to a patient already suffering from the disease in an amount sufficient to eliminate or at least partially alleviate the symptoms of the disease and its complications. Effective doses will vary depending on the condition of the disease to be treated and the judgment of the attending physician based on factors such as the severity of the disease, the age, weight and general condition of the patient.

  The composition to be administered to the patient can be in the form of the pharmaceutical composition described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged and used as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparation is typically between 3 and 11, more preferably 5 to 9, and most preferably 7 to 8. It will be appreciated that pharmaceutical salts are formed by appropriate use of the excipients, carriers or stabilizers described above.

  The therapeutic dosage of the T-type calcium channel antagonist used in the methods described herein will depend on, for example, the particular use in which the treatment is made, the manner of administration of the compound, the health status of the patient and the judgment of the prescribing physician. It can change. The ratio or concentration of a compound of the invention in a pharmaceutical composition can vary depending on many factors such as dosage, chemical properties (eg, hydrophobicity) and route of administration. For example, a T-type calcium channel antagonist can be provided as an aqueous physiological buffer solution containing about 0.1 to about 10% (w / v) of the compound for parenteral administration. Some typical dose ranges are from about 1 μg / kg to about 1 g / kg body weight per day. In some embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg body weight per day. Dosage depends on factors such as the type and degree of progression of the disease or disorder, the overall health status of the particular patient, the relative biological effectiveness of the selected compound, the composition of the excipients and the route of administration. Have a tendency to depend. Effective doses can be extrapolated from dose-response curves derived from in vitro or in vivo model test systems.

  The invention will be further described in the following examples, which do not limit the scope of the invention as defined in the claims.

Treatment of patients with Angelman Syndrome Intended for a patient with Angelman syndrome characterized by mental disability and frequent irregular behavior (eg, seizures, hand flapping). Patients are administered a T-type calcium channel antagonist provided herein (eg, mibefradil, efonidipine, TTL-1177, nickel, etc.) in a therapeutically effective amount, alone or in combination with other therapies. Following administration of a sufficient dosage of a T-type calcium channel antagonist (eg, after a single dose or after a series of doses), the patient's brain activity is monitored (eg, using electroencephalography (EEG)). The patient's brain activity shows a marked improvement in brain activity characteristic of Angelman syndrome compared to the patient's brain activity prior to treatment with a T-type calcium channel antagonist. Patients may also show improvements in other symptoms including intellectual and developmental disorders, sleep disorders, seizures and jerky behavior.

In Vivo Studies of Angelman Model Mice Three studies were conducted separately to determine the effects of two compounds, mibefradil and MK-8998, on behavioral and physiological defects and wrinkles in Angelman syndrome model mice. The tests were audiogenic habit test (Test 1, Tl), exercise / behavior test battery (Test 2, T2) and long-term potentiation measurement (LTP, Test 3, T3). Completion of each test provided a quantitative result of the mibefradil or MK-8998 effect in this test. In all three trials, mice were divided into two separate groups. That is, as shown in Table 2 below, group 1 for test 1 and group 2 for tests 2 and 3, each group comprising six subgroups a to f.

In all behavioral and electrophysiological experiments, female Ube3am ⁻ / p ⁺ KO mice (ie, AS mice) and wild type males were mated and heterozygous AS with a background of F1 hybrid 129S2-C57BL / 6. Mouse littermate controls were made. In the sputum test, AS mice are mated with 129S2 / SvPasCrl background controls. In total, two groups of mice are required: group 1 for sputum testing (Table 2, groups 1a-f), and group 2 for behavioral testing batteries and electrophysiological testing (Table 2, groups 2a-f). f). Each group consists of six subgroups, including two control subgroups: wild type (a) and AS mutant (b) mice fed with a standard diet, and diet fed with a drug 4 Two subgroups (mibefradil (cd) and MK-8998 (ef) groups, two different concentrations each). The number of mice used in the test / subgroup is shown in Table 2, 5-7 mice / subgroup for the electrophysiological test, 10 mice / subgroup for the sputum test and 12-15 for the behavioral test battery Range of mice / subgroups. Adult male and female mice (<8 weeks of age) were used in all experiments.

  Mibefradil and MK-8998 were tested at a variety of different doses (eg, 10 mg / kg, 30 mg / kg, 40 mg / kg, 60 mg / kg) based on the assumption that the mice eat at 5 g diet / day. Both drugs began to be administered with food after weaning the pups (P21, subgroups cf). Control mice (subgroups a and b) were fed a standard diet.

Effects of mibefradil and MK-8998 on wrinkles in an Angelman syndrome (AS) mouse model (Study 1)
AS mutant mice, wild type and AS mutant mice were treated with standard diet (groups Ia and Ib) or diet supplemented with drugs (groups Ic-f) from day 21 after birth (P21) to adulthood. ) Was given and maintained. At 8 weeks of age (P56), all mice were tested for the occurrence of audiogenic seizures. The results of this study were expressed as the number (or%) of mice / subgroups that experienced epilepsy. Most (> 90%) AS mutant mice (group Ib) fed standard diet were expected to experience epilepsy. In contrast, wild-type mice (group Ia) fed standard diet were not expected to show seizures (less than 15%) (eg Silva-santos et al., J. Clin. Invest., 2015, 125 (5), 2069-2076). A summary of the protocol for trial 1 is given below:

1. Seizure evaluation: (129S2 / SvPasCrl × AS mouse)
a. 9 mice / group b. Six groups (as follows):
i. Vehicle: wild type ii. Mibefradil (forced intake by oral gastric tube)
1. 10 mg / kg
2. 40 mg / kg
iii. MK-8998 (forced ingestion by oral gastric tube)
1. 10 mg / kg
2. 30 mg / kg
3. 60mg / kg
c. Treatment schedule: Baseline sputum test → start treatment for 7 days, P56 → sputum test d. Results:% of mice experiencing audiogenic sputum

  First, mice were subjected to sputum tests prior to treatment with mibefradil or MK-8998. Mice were then treated with mibefradil or MK-8998 by gavage via oral gavage for 7 days before being subjected to sputum testing. Mice were dosed as follows: Group 1: Mibefradil 10 mg / kg and 40 mg / kg; MK-8998: 10 mg / kg and 30 mg / kg. Group 2: Mibefradil 10 mg / kg and 40 mg / kg; MK-8998: 10 mg / kg and 30 mg / kg. The results of the audiogenic habit test are shown in FIG.

  As a result, unexpectedly, MK-8998 eliminates seizures in a dose-dependent manner, indicating that seizures are completely eliminated in about 80% of animals that received 60 mg / kg treatment. However, the data shown in FIG. 1 does not describe the apparent response, and FIG. 1 only records the number of animals without seizures and does not indicate that the treatment was ineffective. Should be noted. Although there was a reduction in the number of seizures or the severity of seizures in animals, seizures are still recorded in the data shown in FIG.

  It is expected that seizure-prone animals that are treated with mibefradil or MK-8998 or other T-type calcium channel inhibitors will show reduced seizure frequency or reduced seizure severity, or both.

Test 2: Effect of mibefradil and MK-8998 on behavioral defects in AS mice AS mutant mice show a strong phenotype (Silva-santos et al., J. Clin. Invest., 2015, 125 (5), 2069 -2076), battery of established motor coordination and behavioral tests including rotarod, open field, marble cover, nesting and forced swimming tests. In this test battery, a group of 12-15 mice / subgroup (6 subgroups, see Table 2) is tested as a group of 72-90 mice. Due to breeding and testing capacity limitations, this group is divided into two batches of 7-8 mice / subgroup (42-48 mice / batch). Both batches of mice are subjected to the same exercise / behavioral test battery and the results from both groups are combined to reach statistical power. After each test (eg rotarod), quantifiable results (measurements) are obtained for each subgroup. A summary of the protocol for Test 2 is shown below:

2. Behavioral evaluation: heterozygous AS mice (male wild type x female Ube3am− / p + KO mice)
a. 12-15 mice / group b. Six groups (as follows):
i. Vehicle: wild type ii. Vehicle: heterozygous AS mouse iii. Mibefradil 10 mg / kg
2. 40 mg / kg
iv. MK-8998
1. 30 mg / kg
2. 60mg / kg
c. Treatment schedule: Complete two batches of tests in a 6 week study period.
d. Evaluation and results:
i. Acceleration Rotor Rod: Latency (s)
ii. Marble obscuration: number of marbles not obscured iii. Open field test: Path length (m)
iv. Nesting: Materials used (%)
v. Forced swimming test: Floating time (%).

Test 3: Effect of mibefradil and MK-8998 on synaptic plasticity defects in AS mice Next, the effect of mibefradil and MK-8998 on long-term potentiation (LTP) defects in AS mutant mice is tested. In this experiment, mice subjected to Test 2 can be used. LTP is measured in 5-7 mice / subgroup. If necessary, the number of mice can be increased (eg, from a second batch of mice). A summary of the protocol for Trial 3 is shown below:

3. Long-term potentiation evaluation: heterozygous AS mice (male wild type x female Ube3am− / p + KO mice)
a. 6-7 mice / group b. Six groups (as follows):
i. Vehicle: wild type ii. Vehicle: heterozygous AS mouse iii. Mibefradil 10 mg / kg
2. 40 mg / kg
iv. MK-8998
1. 30 mg / kg
2. 60mg / kg
c. Treatment schedule: 2 mice per day for a total of 5 weeks d. result:
i. LTP induction and expression in wild-type mice (% of baseline)
ii. Rescue potential of drug-induced LTP deficiency in AS mice (% of baseline)

Other Embodiments Although the present invention has been described in connection with the “detailed description of the invention”, the above description is intended to be illustrative only and is intended to limit the scope of the invention. Rather, it is to be understood that the scope of the present invention is defined by the appended claims. A number of embodiments of the invention have been described. However, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other aspects, advantages, embodiments and modifications are within the scope of the following claims.

Claims (59)

  A method of treating Angelman syndrome or Prader-Willi syndrome, comprising administering a therapeutically effective amount of a T-type calcium channel antagonist to a subject in need of such treatment.   2. The method of claim 1, wherein the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.   The method of claim 1 or 2, wherein the T-type calcium channel antagonist is a small molecule.   The method according to claim 1 or 2, wherein the T-type calcium channel antagonist is an antibody.   4. The method of claims 1 to 3, wherein the T-type calcium channel antagonist is siRNA. 6. The method of any one of claims 1-5, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.1. 6. The method of any one of claims 1-5, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.2. 6. The method of any one of claims 1-5, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.3.   9. The T-type calcium channel antagonist antagonizes T-type calcium channels in the cell when the membrane potential of the cell is in the range of about −60 mV to about −30 mV, for example about −40 mV. The method according to claim 1.   The T-type calcium channel antagonist is mibefradil, MK-8998, diltiazem, nifedipine, nitrendipine, nimodipine, nildipine, nigurdipine, nicardipine, nisoldipine, amlodipine, felodipine, isradipine, riocidin, galopamil, verapamil, thiapamil, pimozide, NC 0396, TTL-1177, anandamide, pimozide, penfluridol, clopimozide, flupirylene, haloperidol, droperidol, benperidol, triperidol, melperone, remperone, azaperone, domperidone, anthrafenine, aripiperazole, ciprofloxacin, dapiprazole, dropropridine, Etoperidone, itraconazole, ketoconazole, levodo Propidine, mepiprazole, naphthopidyl, nefazodone, niaprazine, oxypertin, posaconazole, trazodone, urapidil, vesnarinone, manidipine, nilvadipine, benidipine, efonidipine, flunarizine, anandamide, romeridine, phenytoin, zonisamide, N-9203, roll, U-9203, roll , TTA-A2, TTA-A8, TTA-P1, 4-aminomethyl-4-fluoropiperidine (TTA-P2), TTA-Q3, TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944, fence succinimide, mesuximide, desmethylmethosuccimide, efonidipine, trimetadione, dimetadione, ABT-639, TTL-1177, KYSO 044, nickel and Kurutokishin, and is selected from the group consisting of A method according to any one of claims 1 to 9.   1 1. The method of any one of claims 1 to 10, wherein the T-type calcium channel antagonist is selected from the group consisting of mibefradil, MK-8998, efonidipine, TTL-1177 and nickel, and combinations thereof.   12. The method according to any one of claims 1 to 11, wherein the T-type calcium channel antagonist is mibefradil or MK-8998.   The method according to any one of claims 1 to 12, wherein the disease is Angelman syndrome.   The method according to any one of claims 1 to 12, wherein the disease is Prader-Willi syndrome.   15. The method according to any one of claims 1 to 14, wherein the treatment comprises reducing or ameliorating at least one neurological symptom in the subject.   16. The method of claim 15, wherein the neurological symptoms comprise one or more of hyperactivity, abnormal sleep patterns, seizures, dystonia and ataxia.   17. The method of any one of claims 1 to 16, wherein the treatment comprises reducing the frequency of seizures in the subject.   18. The method according to any one of claims 1 to 17, wherein the treatment comprises reducing the severity of the subject's seizures.   19. The method of any one of claims 1 to 18, wherein the treatment comprises reducing the frequency of dystonia in the subject.   20. The method of any one of claims 1 to 19, wherein the treatment comprises reducing the severity of dystonia in the subject.   21. A method according to any one of the preceding claims, wherein the treatment comprises reducing the frequency of ataxia in the subject.   The method of any one of claims 1 to 21, wherein the treatment comprises reducing the severity of ataxia in the subject.   23. A method according to any one of claims 1 to 22, wherein the treatment comprises improving cognition or reducing cognitive impairment in the subject.   24. The method of any one of claims 1 to 23, wherein the treatment comprises improving memory or reducing memory impairment in the subject.   25. A method according to any one of claims 1 to 24, wherein the treatment comprises improving attention or reducing attention deficit in the subject.   26. A method according to any one of claims 1 to 25, wherein the treatment comprises reducing obesity in the subject.   27. The method of any one of claims 1-26, wherein the treatment comprises reducing the amount or rate of weight gain of the subject.   28. The method of any one of claims 1-27, wherein the treatment comprises reducing the subject's weight.   29. A method according to any one of claims 1 to 28, wherein the treatment comprises reducing hunger or increasing satiety in the subject.   30. The method of any one of claims 1 to 29, wherein the selective T-type calcium channel modulator substantially crosses the blood brain barrier.   30. The method of any one of claims 1 to 29, wherein the selective T-type calcium channel modulator does not substantially cross the blood brain barrier.   32. The method of any one of claims 1-31, further comprising administering an additional therapeutic agent to the subject.   35. The method of claim 32, wherein the additional therapeutic agent is a further T-type calcium channel inhibitor.   35. The method of claim 32, wherein the additional therapeutic agent is an anticonvulsant.   35. The method of any one of claims 1-34, further comprising performing a further therapy selected from the group consisting of physical therapy, occupational therapy, communication therapy and behavioral therapy.   A method of treating a disease or disorder associated with CaMKII autophosphorylation in a subject in need thereof, comprising administering to said subject a T-type calcium channel antagonist.   40. The method of claim 36, wherein the disease or disorder is Angelman syndrome.   40. The method of claim 36, wherein the disease or disorder is Prader-Willi syndrome.   39. The method of any one of claims 36 to 38, wherein the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.   40. The method of any one of claims 36 to 39, wherein the T-type calcium channel antagonist is a small molecule.   41. The method of any one of claims 36 to 40, wherein the T-type calcium channel antagonist is an antibody.   42. The method of any one of claims 36 to 41, wherein the T-type calcium channel antagonist is an siRNA. 43. The method of any one of claims 36 to 42, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.1. 43. The method of any one of claims 36 to 42, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.2. 43. The method of any one of claims 36 to 42, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.3.   44. The T-type calcium channel antagonist antagonizes T-type calcium channels in the cell when the membrane potential of the cell is in the range of about −60 mV to about −30 mV, for example about −40 mV. The method according to claim 1.   A method of treating obesity in a subject in need thereof, comprising administering to said subject a T-type calcium channel antagonist.   48. The method of claim 47, wherein the treatment comprises reducing the subject's weight.   49. The method of claim 47 or 48, wherein the treatment comprises reducing the amount or rate of weight gain of the subject.   50. The method of any one of claims 47 to 49, wherein the treatment comprises reducing the subject's weight.   51. The method according to any one of claims 47 to 50, wherein the treatment comprises reducing hunger or increasing satiety in the subject.   52. The method of any one of claims 47 to 51, wherein the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.   53. The method of any one of claims 47 to 52, wherein the T-type calcium channel antagonist is a small molecule.   53. The method of any one of claims 47 to 52, wherein the T-type calcium channel antagonist is an antibody.   53. The method of any one of claims 47 to 52, wherein the T-type calcium channel antagonist is an siRNA. 56. The method of any one of claims 47 to 55, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.1. 56. The method of any one of claims 47 to 55, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.2. 56. The method of any one of claims 47 to 55, wherein the T-type calcium channel antagonist selectively targets Ca _V 3.3. 59. The T-type calcium channel antagonist antagonizes T-type calcium channels in the cell when the membrane potential of the cell is in the range of about −60 mV to about −30 mV, for example about −40 mV. The method according to claim 1.